The effect of the troponin-tropomyosin complex on the binding of myosin subframent-one (S-1) to actin was studied in the presence of different nucleotides as a test of our cooperative binding model developed in collaboration with Dr. Terrell Hill. Our model predicts that both the equilbrium constant between the weak binding form and the strong binding form of actin, and the tropomyosin interaction term should be intrinsic properties of the regulated actin filament. We found that this was the case, thereby confirming our model. In addition, we found that, in the absence of calcium, troponin- tropomyosin inhibits the binding of S-1. ADP (and S-1 alone) to actin much more than the binding of S-1. AMP-PNP. This shows that the nucleotide bound to S-1 determines how strongly troponin-tropomyosin is able to inhibit the binding of the S-1 to actin. In fact, inhibition is completely absent when the bound nucleotide is ATP. (See project no. Z01 HL 00417-02 LCB). These results also suggest that troponin-gropomyosin may inhibit the actomyosin ATPase activity by preventing the rotation of the cross-bridge from the 90 degree (ATP) state to the 45 degree (ADP) state, thereby inhibiting the release of Pi.